Mechanism of xihuang pill inhibition of SLC7A11 deubiquitination through downregulation of the OTUB1 to promote ferroptosis in triple-negative breast cancer.

Ethnopharmacological Relevance: Triple-negative breast cancer (TNBC), a highly aggressive subtype of breast cancer (BC), is more sensitive to ferroptosis than other subtypes. Xihuang pill (XHP) has been extensively used in Chinese clinical practice as an adjuvant therapy for TNBC. However, the underlying mechanisms of XHP in TNBC treatment remain incompletely elucidated.

Aim Of The Study: This study aimed to investigate the anti-TNBC activity of XHP and further clarify its underlying molecular mechanism.

Materials And Methods: An integrated strategy combining network pharmacology with UHPLC-QqQ-MS-based metabolomics was used to identify the active compounds and genes related to XHP that exert anti-TNBC effects by modulating ferroptosis-related protein ubiquitination modifications. In vivo, the therapeutic effects of XHP on TNBC were investigated via a subcutaneous xenograft model. In vitro, we evaluated the effects of XHP on the cell proliferative capacity following DCFH-DA, JC-1, and C11-BODIPY probes, transmission electron microscopy, Fe, MDA, and GSH assay kits were used to detect ferroptosis-related indicators. Potential mechanisms were assessed using western blotting, immunofluorescence, immunohistochemistry, and ubiquitination assays.

Results: OTUB1, SLC7A11, SLC3A2, and GPX4 were identified by network pharmacology in combination with UHPLC-QqQ-MS-based metabolomics as the key targets by which XHP exerts anti-TNBC effects by modulating ferroptosis-related protein ubiquitination modifications. β-boswellic acid, α-boswellic acid, deoxycholic acid, choline, chenodeoxycholic acid, and muscone were identified as key compounds. The experimental results demonstrate that XHP suppresses TNBC cell proliferation, invasion, and migration, safely inhibits tumour growth, with elevated Fe, LPO and ROS levels, and enhances GSH depletion and mitochondrial damage. Our results further confirmed that XHP exerts its anti-TNBC effects by inducing cell ferroptosis, as ferrostatin-1 was found to rescue XHP-induced inhibition of cell proliferation. Mechanistically, XHP promotes the degradation of SLC7A11 via the proteasomal pathway by suppressing OTUB1-mediated deubiquitination, thereby inducing system Xc/GPX4 axis-dependent ferroptosis.

Conclusions: Our study demonstrated that XHP may exert anti-TNBC effects partly by inhibiting OTUB1-mediated deubiquitination of SLC7A11, thereby inducing system Xc/GPX4 axis-dependent ferroptosis. This study provides a foundation for developing potential products from XHP plant materials for the TNBC treatment.